**4.2 Mechanisms of EV entry into the target cell**

When an EV attaches to the target cell surface, it can in some cases activate the cognate receptors without internalization or transfer of the content to the recipient cell via its fusion with the target cell membrane or via endocytosis [84]. Endocytosis is an active process that requires cytoskeletal remodeling dependent on actin dynamics and includes clathrin-dependent endocytosis, phagocytosis and macropinocytosis. The clathrin-dependent endocytosis has been established as cellular entry for EVs based on the experiments with specific inhibitors of this pathway. Additionally, endocytic uptake of EVs can involve lipid rafts, sometimes dependent on caveolin proteins. The size of EVs may be a limiting factor for cellular entry via endocytosis [85]. The EV uptake by phagocytosis was monitored by their high level of accumulation in phagocytic cells and localization into the phagolysosome [86], as well as the identification of the crucial role of the phosphatidylserine binding T-cell immunoglobulin and mucin domain containing (TIM4) receptor for the uptake of exosomes into macrophages [87, 88]. The contribution of macropinocytosis pathway was revealed with studies where exosomal uptake was decreased by the inhibition of cytoskeletal rearrangements that normally lead to membrane ruffles [89], as well as with its promotion caused by the activation of the agonistically acting epidermal growth factor [90].
